The Toxicologist - Society of Toxicology

suggest that TRX possesses novel immunomodulatory properties as a result of its
ability to modulate IL-1β production and/or signalling. In contrast, LF inhibits the
activity of TNF-α. The bioactivity of these molecules following simple topical application
demonstrates that these proteins can access the viable epidermis and may
represent an opportunity for the development of novel anti-inflammatory reagents.
96 PROTEIN ALLERGENICITY AND DIGESTIBILITY:
COMPARISONS OF PEPSIN AND CATHEPSIN.
E. Foster, I. Kimber and R. J. Dearman. University of Manchester, Manchester,
United Kingdom.
An association between protein allergenicity and resistance to pepsin digestion has
been reported previously, however, such is not complete with examples of labile allergens
and resistant non-allergens observed. Given the central role of antigen presenting
cells, such as dendritic cells (DC), in the development of immune and allergic
responses, the stability of allergens to intracellular processing may be more
relevant than resistance to pepsin digestion. We have characterised the expression
by DC of cathepsins (proteolytic enzymes), and compared the proteolytic activity
of the most highly expressed cathepsin with pepsin. Cathepsin expression in bone
marrow-derived DC (BMDC) and in mesenteric lymph node DC derived from
BALB/c strain mice was characterised by flow cytometry. BMDC expressed detectable
levels of cathepsin D, E and S, with cathepsin D being the most highly expressed,
with a similar pattern observed in DC isolated from mesenteric lymph
nodes. Digestion studies revealed that the allergens β-lactoglobulin (BLG), hen egg
lysozme (HEL) and ovalbumin (OVA) were relatively resistant to pepsin, although
bovine serum albumin (BSA) was labile, as were the non-allergens hemoglobin
(HB) and horseradish peroxidise (HRP). In contrast, all 4 allergens were stable to
overnight digestion with cathepsin D, although fragments were observed for BSA.
HB was labile to cathepsin D whereas fragments were recorded for HRP. As intracellular
digestion occurs under reducing conditions, the impact of prior chemical
reduction was explored. Reduced BLG and OVA were labile to pepsin and fragments
were observed following cathepsin D digestion. Generally, allergens were
more stable than non-allergens to digestion by both enzymes and chemical reduction
increased lability. Although a wider range of allergens and non-allergens needs
to be examined, these data suggest that allergenicity correlates more strongly with
resistance to digestion by cathepsin D than with pepsin.
97 INHALATION OF ORTHO-PHTHALALDEHYDE VAPOR
CAUSES SYSTEMIC SENSITIZATION AND ALLERGIC
INFLAMMATION IN THE LYMPH NODES, NASAL
MUCOSA, AND LUNG OF MICE.
V. J. Johnson, W. Wang, K. Fluharty, B. Yucesoy and J. S. Reynolds.
CDC/NIOSH, Morgantown, WV.
Ortho-Phthalaldehyde(OPA) is increasingly being substituted for glutaraldehyde as a
high-level disinfectant for sensitive medical devices. However, toxicological data on
OPA safety is lacking. Safety concerns for glutaraldehyde include acute nasal toxicity
and sensory irritation as well as sensitization leading to occupational rhinitis and
asthma. Since OPA is a dialdehyde like glutaraldehyde and functions as a disinfectant
due to its high reactivity for biological macromolecules, it is reasonable to expect
similar respiratory hazards. Several case reports have been published supporting
this hypothesis showing development of respiratory hypersensitivity reactions
in healthcare workers and patients exposed to OPA. The purpose of this study was
to determine if OPA is a respiratory sensitizer using a murine model. Mice were exposed
via inhalation to OPA vapor 4 hrs/day for 3 days; rested for 11 days; challenged
with OPA vapor 4 hrs/day for 3 days and then sacrificed 24 hrs after the
final exposure. Lungs, nasal mucosa, head-draining lymph nodes (LN) and serum
were collected and processed for cytokine gene expression and flow cytometry.
OPA-specific antibodies were detected in the serum of exposed mice. OPA inhalation
induced a dose dependent increase in LN IL-4 expression and B-cell proliferation.
Importantly, there was a concomitant increase in IgE+ B-cells. Strong Th2 cytokine
expression in the nasal mucosa increased with OPA dose whereas the inverse
was observed for IFNγ expression, a cytokine expression pattern commonly observed
in response to respiratory allergens. Th2 cytokine expression was also increased
in the lung although to a lesser extent. These data demonstrate that OPA
inhalation induces a predominant Th2 cytokine response in the respiratory tract
and draining LN. Importantly, OPA inhalation induced isotype switching to IgE in
the draining LN supporting the development of an allergic immune response.
Overall, this study suggests that inhalation of OPA vapor in mice can induce respiratory
allergy.
20 SOT 2011 ANNUAL MEETING
98 CONTACT SENSITIZING POTENTIAL OF
HEPTACHLOR IN FEMALE BALB/C MICE.
R. P. Frawley 1 , W. Auttachoat 2 , M. J. Smith 2 , R. D. Brown 2 , T. L. Guo 2 , K. L.
White Jr 2 and D. R. Germolec 1 . 1 National Toxicology Program, National Institute of
Environmental Health Sciences, Research Triangle Park, NC and 2 Department of
Pharmacology and Toxicology, Virginia Commonwealth University, Richmond, VA.
Heptachlor (HPT) is a chlorinated cyclodiene insecticide that was used extensively
in the 1950’s through 1980’s in agriculture, and in commercial and domestic buildings.
Banned by the Environmental Protection Agency (EPA) for most uses (except
for fire ant control in underground power cable boxes) since 1988, low levels of
HPT residues persist in the environment. Human exposure can occur through contact
with contaminated soil or building materials; consumption of contaminated
meat, fish or dairy products; or production and application of HPT insecticides.
The objective of this study was to evaluate the sensitization potential of HPT in female
BALB/c mice following dermal exposure using the ICCVAM-validated local
lymph node assay (LLNA) in combination with a measurement of irritancy, and the
mouse ear swelling test (MEST). Sensitization was evaluated at concentrations that
did not induce overt toxicity. HPT was applied to the dorsa of both ears daily for
three days. There was no increase in ear swelling 24 hr following the third application
at 0.2%-5.0%, indicating that HPT is not an irritant. Cervical (auricular)
lymph node cell proliferation was statistically increased (72 hr post-application) at
2% HPT relative to the vehicle control, however, the increase did not reach a sensitization
index (SI) of 3. In the MEST, mice sensitized with 1% and 2% HPT and
challenged with 2% HPT exhibited significant increases in percent ear swelling,
compared to the vehicle irritancy control, at 24 hr, but not at 48 hr, post-challenge.
In summary, based on lack of irritancy and on statistical significance in the LLNA
and MEST, HPT can be characterized as a sensitizer, but not an irritant.
99 HUMAN LINE1 PROMOTER ACTIVITY IS ENHANCED
BY CHEMICAL AND DRUG-INDUCED STRESS IN
HEPG2 CELLS.
N. Terasaki, M. Kajikawa and N. Okada. Department of Biological Science,
Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology,
Yokohama, Japan. Sponsor: J. Sugimoto.
Long interspersed element 1 (LINE1), which is one of retrotransposons, constitutes
17 % of the human genome. 80-100 human LINE1 elements are currently active
and can mobilize into a new location of the genome, resulting in alteration of the
genomic information. Therefore, active LINE1 elements are considered as a sort of
endogenous mutagens. In previous studies, some stresses, for example, heat shock,
gamma radiation, UV irradiation and some agents are reported to induce mobilization
of retroposons. In this study, to investigate widely what chemicals and drugs
have the potential to enhance the LINE1 promoter activity, we established a reporter
gene assay system in HepG2 cells using human LINE1. We cloned the 5’-
UTR of L1.3, which is one of the most active human LINE1s, in the upstream of a
luciferase gene and used it in the assay. The LINE1 promoter activity was measured
at 6 and 24 hours after exposure to compounds. We assessed 106 compounds
which include anticarcinogenic agents, nonsteroidal antiinflammatory drugs
(NSAIDs), hypolipidemic agents, statins and some compounds that induce ER
stress, oxidative stress, mitochondrial dysfunction etc. More than 1.5-fold increase
of the LINE1 promoter activity was observed by the treatment of 9 compounds
(merbarone, benzo(a)pyrene, exo1, iodoacetamide, citrinin, cyclosporine A, fenofibrate,
bezafibrate, diflunisal and salicylamide) at both 6- and 24-hour points. Other
12 compounds also elevated the LINE1 promoter activity by 1.2- to 1.5-fold.
These results suggest that various toxicants and drug stresses might have the potential
to cause genomic mutations in a human body by inducing LINE1 mobilization.
Therefore, we should keep in mind the possibility of activation of retrotransposons
during drug discovery processes.
100 IN UTERO BISPHENOL A EXPOSURE ALTERS
METASTABLE EPIALLELE AND GLOBAL DNA
METHYLATION PATTERNS IN MOUSE OFFSPRING.
M. S. Nahar, C. Weinhouse, O. S. Anderson, T. R. Jones, S. A. Liberman, L. S.
Rozek and D. C. Dolinoy. Environmental Health Science, University of Michigan,
Ann Arbor, MI.
Genetically identical individuals such as monozygotic twins and inbred mice often
display phenotypic discordance, even after controlling for environment. Therefore,
epigenetic plasticity has been proposed to play a role in the diverse phenotypes of
individuals. Metastable epialleles variably express loci in genetically identical individuals
due to epigenetic marks established early in development. Strikingly, these